The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
Receptor protein tyrosine kinases (“RPTKs”) include a large and diverse family of enzymes. The RPTK family contains multiple subfamilies, one of which is the fibroblast growth factor receptor (FGFR) subfamily. Another subfamily is the type III receptor tyrosine kinase (RTK) subfamily whose members include platelet-derived growth factor receptors a and β (“PDGFR α” and “PDGFR β”), macrophage colony-stimulating factor receptor (M-CSFR), c-kit (also referred to as SCF receptor (“SCFR”)) and the flt3 receptor. The members of this RTK subfamily contain five immunoglobulin-like (Ig) domains in their extracellular ligand binding domains followed by a single transmembrane domain and a cytoplasmic tyrosine kinase domain interrupted by a large kinase-insert. For a review of RPTKs, see Schlessinger and Ullrich, 1992, Neuron 9: 383–391; for a review describing the FGFR subfamily, see Givol and Yayon, 1992, FASEB J. 6 (15): 3362–3369.
All RPTKs enzymatically transfer a high energy phosphate from adenosine triphosphate to a tyrosine residue in a target protein. These phosphorylation events regulate certain cellular phenomena in signal transduction processes. Cellular signal transduction processes contain multiple steps that convert an extracellular signal into an intracellular signal. The intracellular signal is then converted into a cellular response. RPTKs are components in many signal transduction processes. Typically, an RPTK regulates the flow of a signal in a particular step in the process by phosphorylating a downstream molecule. This phosphorylation modulates the downstream molecule's activity by turning it either “on” or “off,” causing excessive or deficient signalling by the downstream molecule. Excessive signalling can lead to such abnormalities as uncontrolled cell proliferation, which is characteristic of such disorders as cancer, angiogenesis induced by various tumors, atherosclerosis, and arthritis. Alternatively, cellular proliferation can be induced therapeutically, for example angiogenesis may be used to ameliorate coronary artery disease by inducing collateral vascularization.
Ligand-induced dimerization of RPTKs is an important step in the RPTK-mediated signal transduction process. For review of the importance of dimerization of RPTKs, see Lemmon and Schlessenger, 1994, Trends in Biochem. Sci. 19: 459463; and Ullrich and Schlessenger, 1990, Cell 61:203–212. Some growth factors, for example platelet-derived growth factor (“PDGF”) and stem cell factor (“SCF”), are dimeric molecules that, by themselves, induce dimerization of their specific receptors. In contrast, other growth factors, such as fibroblast growth factors (FGFs), are monomeric molecules that must act in concert with other molecules to induce dimerization of their specific receptors. See Schlessenger et al., 1995, Cell 83: 357–360; Spivak-Kroizman et al., 1994, Cell 79: 1015–1024; Ornitz et al., 1992, Mol. Cell. Biol. 12: 240–247. In particular, FGFs typically function in concert with soluble or cell surface-bound heparin sulfate-containing proteoglycans (HSPGs).
The FGFR subfamily consists of at least 21 structurally related polypeptides, designated FGFR1 through FGFR21, that are expressed in embryonic, fetal, and adult vertebrates. FGFR1 through FGFR4, are known as “high affinity FGFRs,” due to their ability to bind appropriate fibroblast growth factors with a high affinity. These high affinity FGFRs are characterized by an extracellular ligand-binding domain which comprises three immunoglobulin (IG)-like domains (known as D1, D2, and D3), a single transmembrane helix, and a cytoplasmic domain containing tyrosine kinase activity. See Lee et al., 1989, Science 245: 57–60; Jaye et al., 1992, J. Mol. Biol. 227: 840–851; Johnson & Williams, 1993, Adv. Cancer. Res. 60: 1–41. Each of the four high affinity FGFRs binds to a specific subset of FGFs. Ornitz et al., 1996, J. Biol. Chem. 271: 15292–15297.
Naturally occuring variants of the high affinity FGFRs-lacking D1, or D1 and the linker region between D1 and D2 known as the “acid box,” have been identified. These varient FGFRs retain the ability to bind appropriate FGFs with high affinity, suggesting that the D2 and D3 regions are sufficient to confer FGF binding ability and specificity. See Crumley et al., 1991, Oncogene 6: 2255–2262; Dionne et al., 1990, EMBO J. 9: 2685–2692; Johnson and Williams, 1993, Adv. Cancer. Res. 60: 141. In particular, D3 has been shown to play a critical role in the binding specificity of FGFRs. See Bottaro, et al., 1990, J. Biol. Chem. 265: 12767–12770; Miki et al., 1992, Proc. Natl. Acad. Sci. 89: 246–250; Dell et al., 1992, J. Biol. Chem. 267: 21225–21229; Yayon et al., 1992 EMBO J. 11: 1885–1890.
Recently, three dimensional structures of the intracellular catalytic domains of various PTKs have been described in International Publication No. WO 98/07835, U.S. patent application Ser. No. 60/034,168, filed Dec. 19, 1996, and U.S. Pat. No. 5,942,428, issued on Aug. 24, 1999, each of which is hereby incorporated herein by reference in its entirety including all claims, drawings, tables, and figures.
Despite recent advances in the understanding of signal transduction and function of the receptor PTKs and their ligands, there remains a need in the art for the atomic-level characterization and analysis of such molecules, particularly with respect to the design and synthesis of novel and improved therapeutic molecules.